This invention relates generally to systems for culturing cells, and more particularly, to a collagen-coated polystyrene microcarrier bead system which serves as a microcarrier support for the culturing of anchorage-dependent cells.
The use of carrier support systems to facilitate growth of biological cells is long and varied in its history. Early systems for effecting such cell growth in usable quantities have included the well-known petri dish and flasks. Efforts intended to increase the quantity of cell production have resulted in the use of large trays; the overall effect achieved being the increase in the size of the surface on which the biological cells are grown, specifically anchorage-dependent cells. Some of these early cell-growth systems are still in use for applications where small-scale production will suffice, such as in hospital and university research units.
The state of the cell culturing art has continued to develop to the present day where there is an acute need for large scale commercial cell culturing systems which can achieve high rates of production. Ideally, the production rate should be flexible to accommodate batch and single-order production.
At present, the cell culturing system which enjoys utilization in some 90% to 95% of the commercial market is the roller bottle system. Essentially, a roller bottle is a cylindrical container which is arranged to contain a small amount of nutrient media. In operation, the roller bottle is rotated slowly, at about 1 to 3 rpm, whereby the nutrient media is continually caused to wet the entire interior surface of the bottle, on which cell growth is achieved. A plurality of such roller bottles are operated on a roller rack, the specific number thus rotated being responsive to the desired rate of overall cell production.
The remaining 5% to 10% of the commercial large-scale market utilizes microcarrier systems. Although other techniques have been introduced in recent years, including hollow fibers, fiber bundles, and channeled ceramic cores, only microcarrier systems have the potential for scale-up within a given reactor to achieve anchorage-dependent cell growth at commercially advantageous production rates and volumes. Scale-up in this manner is superior to scale-up by replication, as is the case with roller bottles and other known systems. In addition, microcarrier bioreactor systems are well-suited for automation systems and controlled large-scale cultivation of anchorage-dependent cells.
As is evident from Table 1, below, several microcarrier systems are presently commercially available. This table summarizes some of the characteristics of the commercially available microcarriers, including polystyrene, glass-coated polystyrene, and the novel collagen-coated polystyrene microcarriers of the present invention.
TABLE 1 __________________________________________________________________________ BEAD BEAD SURFACE CHEMICAL DENSITY SIZE AREA MANUFACTURER COMPOSITION g/cc REUSABLE .mu.m cm/g AND COUNTRY __________________________________________________________________________ Poly- 1.02 Y 90-210 475 SoloHill, USA Styrene (PS) Glass- to Y 90-210 to SoloHill, USA Coated PS Collagen- 1.04 N 90-210 325 SoloHill, USA Coated PS DEAE 1.03 N 130-220 4-6000 Pharmacia, Swd Dextran Collagen-Coated 1.04 N 100-190 3-5000 Pharmacia, Swd DEAE Dextran Polystyrene 1.05 N 160-300 255 Nunc, Denmark Polystyrene 1.05 N 160-230 -- Lux, USA Collagen-Coated 1.05 N 160-250 -- IBF, France Polyacrylamide Collagen 1.04 N 115-230 3-4000 Hazelton, USA Gelatin Collagen and -- N 150-250 -- Galil, Israel Coated DEAE Variants __________________________________________________________________________
The Swedish-based company, Pharmacia, was the first to introduce the microcarrier bead in the early 1970's. Subsequently, this company has acquired about a 90% share of the microcarrier bead market, worldwide.
The use of microcarrier beads as the microcarrier elements in anchorage-dependent cell production systems requires the availability of bioreactors, support equipment, and a stirring system. The system elements interact with one another to maintain the cell-laden microcarrier beads in suspension in the nutrient media. Much of this type of equipment is commercially available, and the effort to develop and improve bioreactor systems for use with microcarrier beads has intensified.
A problem which has significantly reduced the rate of cell production in existing systems is that of damage to the cells resulting from shear forces. During stirring, turbulence in the nutrient media impose forces upon the cells under development. The cells are so fragile that these forces both damage them and reduce their proliferation. The obvious answer to this problem is to reduce the shear forces by slowing the stirring system. However, if the potency of the stirring system is reduced too far, the microcarrier beads with their attached cells will settle and come into contact with one another and the interior walls of the container. One approach to reducing the stirring forces so that the damage to the cells being cultivated is diminished, is to decrease the density of the microcarrier beads. Microcarrier beads having lower density require less fluid turbulence to maintain them in suspension, thereby also reducing cell damage.
It is, therefore, an object of this invention to provide a simple and economical system for supporting the growth of anchorage dependent cells.
It is another object of this invention to provide a system for supporting the growth of anchorage-dependent cells which can be sterilized by standard techniques.
It is also an object of this invention to provide a microcarrier system for supporting the growth of anchorage-dependent cells.
It is a further object of this invention to provide a microcarrier system for supporting the growth of anchorage dependent cells at flexible production rates, without the need for high initial capital investment.
It is additionally an object of this invention to provide a microcarrier system which has a low density.
It is yet a further object of this invention to provide a low-density microcarrier system which can be sterilized by autoclaving methods.
It is also another object of this invention to provide a microcarrier system wherein product contamination during cell harvesting is minimized.
It is yet an additional object of this invention to provide a microcarrier system for facilitating the growth of anchorage-dependent cells wherein cell recovery is high and cell separation can easily be accomplished using proteolytic enzymes.
It is still another object of this invention to provide a microcarrier system wherein essential production features are independent of the dimensions of the microcarrier system over a broad range.
It is a yet further object of this invention to provide a microcarrier system wherein cell attachment takes place quickly.
It is also a further object of this invention to provide a microcarrier system for facilitating the growth of anchorage-dependent cells wherein absorption of culture media, products, or toxins is inhibited.
It is additionally another object of this invention to provide a microcarrier bead system which can be formed using existing laboratory methods and systems.
A still further object of this invention is to provide a microcarrier bead system which can be produced in a broad range of predetermined bead dimensions.
An additional object of this invention is to provide a microcarrier bead system wherein the manufacturing size distribution for a predetermined bead size is narrower than existing microcarrier products.